Two noncompeting human neutralizing antibodies targeting MPXV B6 show protective effects against orthopoxvirus infections

The recent outbreak of mpox epidemic, caused by monkeypox virus (MPXV), poses a new threat to global public health. Here, we initially assessed the preexisting antibody level to the MPXV B6 protein in vaccinia vaccinees born before the end of the immunization program and then identified two monoclonal antibodies (MAbs), hMB621 and hMB668, targeting distinct epitopes on B6, from one vaccinee. Binding assays demonstrate that both MAbs exhibit broad binding abilities to B6 and its orthologs in vaccinia (VACV), variola (VARV) and cowpox viruses (CPXV). Neutralizing assays reveal that the two MAbs showed potent neutralization against VACV. Animal experiments using a BALB/c female mouse model indicate that the two MAbs showed effective protection against VACV via intraperitoneal injection. Additionally, we determined the complex structure of B6 and hMB668, revealing the structural feature of B6 and the epitope of hMB668. Collectively, our study provides two promising antibody candidates for the treatment of orthopoxvirus infections, including mpox.

• The orientation distribution plot in Figure S4D.
• The 2D class averages in Figure S4B (see below).Only two unique views of the complex seem to be apparent.
• The unmodified sharpened map lacks features expected in a 3.4 Å resolution map.
2) Related to point 1, the authors have built an atomic model using the DeepEMhancer map and used this for refinement.Given that the AI-modified map has features that are not present in the unmodified data, it's not clear to me if this model can be trusted.This probably explain the poor validation scores for the result pdb file compared to models of a similar resolution.

Minor issues
1) The FSC curve shown in Figure S4C never reaches zero.This is indicative of issues with the data, such as the presence of duplicate particles.https://discuss.cryosparc.com/t/why-do-duplicateparticles-appear/12072 2) The authors have performed single-particle image processing using particles with a pixel size of 0.68 Å/pixel.While not strictly incorrect, the resolution of the resulting map is so far away from Nyquist that using such a small pixel size makes no sense.In fact, by binning the data 1.5x, the improved signal-to-noise may improve alignment of particles during refinement.

Recommendations
Despite the major issues with the cryo-EM map and model, the overall interpretation of the structural data appears to be correct.The resolution of the (unmodified map) is good enough to fit the AlphaFold model and make an educated guess about the epitope-paratope residues.The way I see it, the authors have two options on how to proceed.
Option 1: Be more forthcoming with the limitations of the current data set.
• The authors should carry out orientation diagnostics of their data and include this as a supplementary figure.This analysis can be performed in the latest version of CryoSPARC.https://guide.cryosparc.com/processing-data/all-job-types-in-cryosparc/utilities/job-orientationdiagnostics • The authors should show example density for the modified and unmodified regions of the map with the fitted pdb.
• Remove domain-level RMSD values.Given the limited resolution, these values are not reliable.Description of movements between domains would be ok to describe at this resolution.
• I would strongly advise using unmodified maps for refinement.There are several nice tools for flexibly fitting models (such as your AlphaFold coordinates) into lower resolution maps (e.g.Namdinator https://namdinator.au.dk/).Option 2: Obtain a higher-quality experimental structure.
• I appreciate this is probably not feasible given the time and effort required to generate a structure, but there are several ways to alleviate the strong preferred orientation seen in your data.Collecting a tilted data set would be the most straightforward.I can think of one example of a similar sized complex on graphene oxide that was determined from tilted data: https://www.nature.com/articles/s41467-023-38509-2#Sec18 Reviewer #2 (Remarks to the Author): This manuscript describes the isolation, expression, and characterization of two anti-B5 human monoclonal antibodies from B-cells from a previously vaccinated person.One (hMB668) competes with binding to mpox-B5 of a previously published monoclonal (MAb 8AH8AL,ref 26).A cryo-EM structure of hMB668 bound to the first two SCR's of mpox-B5 is also presented and data from that may explain the slightly lower binding of this MAb to the VARV-and CPXV-B5 proteins compared to the MPXV-and VACV-B5 proteins.Authors provide evidence that the other human Mab isolated (hMB621) bound to SCR3-SCR4, but Cryo-EM structure of this complex could not be obtained.
Both MAb showed EV neutralizing activity in the presence of complement and when given 4 hr before and 4 hr after challenge, showed protection of mice in an intranasal mouse challenge model with VACV.
B5 is known to be an important target of a protective immune response and this paper supports this and provides additional important characterization of B5 and human MAbs that may some day be used as part of a cocktail of monoclonal antibodies that could be used for the treatment or prevention of orthopoxvirus infections.
Major points for authors to address: What subtype IgG are these MAbs?Do these antibodies inhibit comet plaque formation?Need to include the genebank names of the genes expressed.Is the sequence of those expressed proteins the ones shown in Supplementary figure 1A? Minor points 1. Abstract, line 33.I do not think the data shows the molecular basis of the neutralization and protection of the MAb targeting the B6.At best the cryo EM shows the site of binding /interaction with B6.
2. Line 38/39.Would include the word complement since that is a key feature of the neutralization activity.
3. Lines 56.Statements not entirely correct.In U.S., the Jynneos (MVA) vaccine is approved as a vaccine to prevent mpox.4. Lines 64 & 67.While there had been limited clinical data on its effectiveness of MVA to protect against mpox, since the outbreak there has been a lot more data.Authors should also cite more studies that show better effectiveness than the one study they cite.The manuscript by Zhao et al describes two monoclonal antibodies (mAbs), hMB621 and hMB668, targeting distinct epitopes on the MPXV B6 protein.Using binding assays, the authors demonstrate that these mAbs can bind to B6 orthologs in vaccinia, variola, and cowpox viruses.These data are further complemented by neutralization and in vivo protection data using vaccinia.Finally, the authors determined the cryo-EM structure of B6 and hMB668, revealing the molecular basis of the neutralization and protection of the mAb targeting B6.It is this latter aspect of the work that I will focus my review on.
Given the public health threat posed by MPXV, the results of this study are important.Moreover, the manuscript is well-written, and the results are straightforward.However, there are serious issues with the cryo-EM data and its overinterpretation.As such, I cannot, recommend the publication of this data in its current form as it would not stand up to the scrutiny of the structural biology community.

Major Concerns
1) The authors utilized DeepEMhancer, a deep learning approach designed for automatic postprocessing of cryo-EM maps, to modify their map.While such methods are not inherently frowned upon in the cryo-EM community, in this particular instance, the resulting modified map introduces features that are not present in the unmodified data.This essentially masks issues with the map, specifically its high anisotropy.This is evident through the following: • The orientation distribution plot in Figure S4D.
• The 2D class averages in Figure S4B (see below).Only two unique views of the complex seem to be apparent.
• The unmodified sharpened map lacks features expected in a 3.4 Å resolution map.
Response: Thanks for your constructive suggestions.Following your suggestions, we reprocessed the cryo-EM data by removing the duplicates and finally acquired an unmodified map at a resolution of 3.46 Å.This map was used for refinement.We also performed orientation diagnostics and prepared the new Figure 5 and Supplementary Figure 4, shown as follows.
Figure 5 Supplementary Figure 4 2) Related to point 1, the authors have built an atomic model using the DeepEMhancer map and used this for refinement.Given that the AI-modified map has features that are not present in the unmodified data, it's not clear to me if this model can be trusted.This probably explain the poor validation scores for the result pdb file compared to models of a similar resolution.
Response: Using the unmodified map for refinement resulted in an improved validation score of 16 for the PDB file, compared to the previous score of 26.

Minor issues
1) The FSC curve shown in Figure S4C never reaches zero.This is indicative of issues with the data, such as the presence of duplicate particles.https://discuss.cryosparc.com/t/why-do-duplicate-

particles-appear/12072
Response: By reprocessing the cryo-EM data, including the removal of duplicates, the resulting FSC curve reached zero, as shown in the new Supplementary Figure 4c.
2) The authors have performed single-particle image processing using particles with a pixel size of 0.68 Å/pixel.While not strictly incorrect, the resolution of the resulting map is so far away from Nyquist that using such a small pixel size makes no sense.In fact, by binning the data 1.5x, the improved signal-to-noise may improve alignment of particles during refinement.
Response: Thanks for your good suggestion.However, due to the limited cryo-EM resources, we are currently unable to reacquire the data.We greatly appreciate your suggestion and will explore it in future studies.

Recommendations
Despite the major issues with the cryo-EM map and model, the overall interpretation of the structural data appears to be correct.The resolution of the (unmodified map) is good enough to fit the AlphaFold model and make an educated guess about the epitope-paratope residues.The way I see it, the authors have two options on how to proceed.
Option 1: Be more forthcoming with the limitations of the current data set.
• The authors should carry out orientation diagnostics of their data and include this as a supplementary figure.This analysis can be performed in the latest version of CryoSPARC.https://guide.cryosparc.com/processing-data/all-job-types-in-cryosparc/utilities/job-orientationdiagnosticsResponse: Thanks for your valuable suggestion.We performed the orientation diagnostics using the CryoSPARC v4.2.1, due to the error issues with our rented server for the latest CryoSPARC v4.4.
The outcomes have been included in the new Supplementary Figure 4d and f, shown as follows.
• The authors should show example density for the modified and unmodified regions of the map with the fitted pdb.
Response: We have prepared the following figure to show the modified and unmodified map with the corresponding fitted pdb.The modified map is sharpened compared to the unmodified map.
• Remove domain-level RMSD values.Given the limited resolution, these values are not reliable.
Description of movements between domains would be ok to describe at this resolution.
Response: We have revised the description about the domain-level RMSD in the manuscript.
• I would strongly advise using unmodified maps for refinement.There are several nice tools for flexibly fitting models (such as your AlphaFold coordinates) into lower resolution maps (e.g. Namdinator https://namdinator.au.dk/).

Response: Done.
Option 2: Obtain a higher-quality experimental structure.
• I appreciate this is probably not feasible given the time and effort required to generate a structure, but there are several ways to alleviate the strong preferred orientation seen in your data.Collecting a tilted data set would be the most straightforward.I can think of one example of a similar sized complex on graphene oxide that was determined from tilted data: https://www.nature.com/articles/s41467-023-38509-2#Sec18 Response: Thanks for your understanding and good suggestion.However, due to the limited cryo-EM resources, we are currently unable to reacquire the data.We appreciate your and will explore it in future studies.

Reviewer #2 (Remarks to the Author):
This manuscript describes the isolation, expression, and characterization of two anti-B5 human monoclonal antibodies from B-cells from a previously vaccinated person.One (hMB668) competes with binding to mpox-B5 of a previously published monoclonal (MAb 8AH8AL,ref 26).A cryo-EM structure of hMB668 bound to the first two SCR's of mpox-B5 is also presented and data from that may explain the slightly lower binding of this MAb to the VARV-and CPXV-B5 proteins compared to the MPXV-and VACV-B5 proteins.Authors provide evidence that the other human Mab isolated (hMB621) bound to SCR3-SCR4, but Cryo-EM structure of this complex could not be obtained.
Both MAb showed EV neutralizing activity in the presence of complement and when given 4 hr before and 4 hr after challenge, showed protection of mice in an intranasal mouse challenge model with VACV.
B5 is known to be an important target of a protective immune response and this paper supports this and provides additional important characterization of B5 and human MAbs that may some day be used as part of a cocktail of monoclonal antibodies that could be used for the treatment or prevention of orthopoxvirus infections.
Major points for authors to address: 1. What subtype IgG are these MAbs?
Response: In the process of the MAbs isolation, we used the anti-IgG antibody to sort the IgG + memory B cells.Thus, we can not tell the subtype of the original IgG.However, we constructed the isolated MAbs as IgG1 subtype to evaluate their activities in vitro and in vivo.

Do these antibodies inhibit comet plaque formation?
Response: Thanks for your comment.In this study, we utilized the Western Reserve (WR) strain of VACV to evaluate the antiviral effectiveness of hMB621 and hMB668.This strain can form round plaques but not comet-shaped ones.Several published studies have shown that the IHD-J strain of VACV can form comet-shaped plaques.However, due to the inaccessibility of the strain, we were unable to evaluate the inhibitor efficacy of the MAbs.Nevertheless, the MAb 8AH8AL showed a reduction in comet plaque formation 1 .Therefore, we speculate that the hMB621 and hMB668, especially hMB668, could inhibit comet plaque formation similar to 8AH8AL, as they recognize a similar epitope.We will explore it in future studies.
3. Need to include the genebank names of the genes expressed.Is the sequence of those expressed proteins the ones shown in Supplementary figure 1A?
Response: The sequences of the expressed MPXV B6 protein and its orthologs are the ones shown in Supplementary Figure 1A.We have included the isolated strains and the accession numbers of these proteins in the Materials and Methods section and the legend of Supplementary Figure 1.The MPXV B6 in the original manuscript is from MPXV_USA_2022_MA001 strain, belonging to clade II.We also added MPXV B6 sequence from the representative strain (Zaire-96-I-16) of clade I and revised Supplementary Figure 1a and b.The new Supplementary Figure 1 is shown below.

5.
Line 78.Instead of clinic, would say in humans.6. Line 110.Would not call B5 a poorly characterized protein.This has been an extensively studied orthopoxvirus protein 7.Line 115.Would end sentence with "in the presence of complement." 8. Line 116.Sentence should read, Moreover, in an intranasal VACV mouse challenge model, the two MAbs exhibited effective protection when given via intraperitoneal (i.p.) injection.9. Line 170.Title should read: Neutralization of antibodies in vitro against VACV infection is complement dependent 10.Line 181.Would include what a 10 mg/kg dose represents in mice to better compare to published data in ref 25 which used humanized MAb at 90 ug of purified Mab.11.Lines 190/191.Given the nature of the challenge model (likely non-lethal challenge dose and other factors), better today, data shows that these MAbs can effectively treat VACV infections in this mouse model.12. Line 193.More correct title: Molecular basis of antibodies binding to MPXV B6 Figure 5 as shown below.The colors have been indicated in the legend.
13. Line 282.Better to say, "Since we could not work with live MPXV .." 14.Line 287.Since neutralization is complement dependent, not sure these can be called "neutralizing epitopes".15.Line 293.Similar to the prior comment, not sure one can say neutralize.How many times was the challenge experiment performed.Seems like it was just once with 5-mice per group?21.Supplementary figure 1. Need to include gene bank name of proteins being used.Also given interest in Clade 1 and Clade 2 MPXV, would include a representative B5 sequence from each.22. Supplementary Figure 5. None of the colors used in the figure are explained.For example, panel H and I. What is the SCR and what is the antibody?